Adventitious roots were inoculated into the bioreactor
by filling it with 1.5 dm3 of corresponding liquid medium
(see above) and 20.5 g of roots. After one month of
cultivation, 0.5 dm3 of fresh medium was added up to a
total volume of 2.0 dm3 in the bioreactor tank. The
cultivation lasted 8 weeks. Adventitious roots grew
slower than the suspension; the cultivation period was
therefore doubled, following the double subcultivation
period of standard cultivation in Erlenmeyer flasks.
For detection of ginsenosides plant material was homogenized
and extracted with 7 cm3 methanol g-1 (f.m.) for
5 d at room temperature. The sample was then filtered
and evaporated to dryness under vacuum. The residue of
the extract was redissolved in distilled water and
partitioned with diethyl-ether, and twice in n-BuOH
saturated with water. The n-BuOH layer was
concentrated in vacuo to obtain a crude saponin fraction
(Sanada 1974). HPLC analysis of the n-BuOH soluble
fraction was then used for the identification and
quantification of ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf
and Rg1. HPLC analyses were performed in a system
consisting of two high pressure pumps (SDS 020 a
SDS 030, DeltaChrom, Watrex, Prague, Czech Republic)
with a mixer and PDA detector (MD 1510, Jasco, Tokyo,
Japan); the stainless steel column (250 × 4 mm) packed
with reverse phase Si-C18, 7 μm (Biospher, Prague,
Czech Republic); flow-rate 1 cm3 min-1. The injection
volume was set up at 0.02 cm3 in the autosampler
(AS300, TSP, San Jose, USA). Eluents: A - 15 %
acetonitrile and water, B - 100 % acetonitrile. Gradient
elution profile: 0 - 40 min, 0 - 35 % B; 40 - 45 min, 35 %
B. The peaks were monitored by UV detection at 203 nm
(Soldati and Sticher 1980, Pietta et al. 1986, Petersen and
Palmqvist 1990). Each ginsenoside was identified by
comparing its retention time and UV spectra with
authentic ginsenosides purchased from Carl Roth GmbH
& Co., Karlsruhe, Germany. The ginsenoside content was
expressed in mg.g-1(f.m.). The presence of ginsenosides
was additionally confirmed by LC-MS.
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